Enriching Chemical Space of Bioactive Scaffolds by New Ring Systems: Benzazocines and Their Metal Complexes as Potential Anticancer Drugs

The search for new scaffolds of medicinal significance combined with molecular shape enhances their innovative potential and continues to attract the attention of researchers. Herein, we report the synthesis, spectroscopic characterization (1H and 13C NMR, UV–vis, IR), ESI-mass spectrometry, and single-crystal X-ray diffraction analysis of a new ring system of medicinal significance, 5,6,7,9-tetrahydro-8H-indolo[3,2-e]benzazocin-8-one, and a series of derived potential ligands (HL1–HL5), as well as ruthenium(II), osmium(II), and copper(II) complexes (1a, 1b, and 2–5). The stability of compounds in 1% DMSO aqueous solutions has been confirmed by 1H NMR and UV–vis spectroscopy measurements. The antiproliferative activity of HL1–HL5 and 1a, 1b, and 2–5 was evaluated by in vitro cytotoxicity tests against four cancer cell lines (LS-174, HCT116, MDA-MB-361, and A549) and one non-cancer cell line (MRC-5). The lead compounds HL5 and its copper(II) complex 5 were 15× and 17×, respectively, more cytotoxic than cisplatin against human colon cancer cell line HCT116. Annexin V-FITC apoptosis assay showed dominant apoptosis inducing potential of both compounds after prolonged treatment (48 h) in HCT116 cells. HL5 and 5 were found to induce a concentration- and time-dependent arrest of cell cycle in colon cancer cell lines. Antiproliferative activity of 5 in 3D multicellular tumor spheroid model of cancer cells (HCT116, LS-174) superior to that of cisplatin was found. Moreover, HL5 and 5 showed notable inhibition potency against glycogen synthase kinases (GSK-3α and GSK-3β), tyrosine-protein kinase (Src), lymphocyte-specific protein-tyrosine kinase (Lck), and cyclin-dependent kinases (Cdk2 and Cdk5) (IC50 = 1.4–6.1 μM), suggesting their multitargeted mode of action as potential anticancer drugs.


■ INTRODUCTION
Rings are favored building blocks of approved drugs and compounds in clinical trials 1−8 but also used in compounds at the early stage of development. 9−11 They determine the electronic distribution, shape, and scaffold flexibility. These electronic and geometric features are often key factors determining molecular properties such as lipophilicity or polarity, which may be responsible for the molecule's reactivity and toxicity. 12 Generally, the number of rings or ring systems (for definition, see ref 1) in drugs is smaller when compared with those in clinical trials. Systematic changes of up to two atoms on existing drug and clinical trial ring systems are expected to lead to future clinical trial scaffolds, which are predicted to cover ∼50% of the novel ring systems entering clinical trials. An innovative drug was defined recently as a drug in which both the scaffold and the molecular shape had not been observed in a previous drug molecule. 3 Recent analyses of all approved drugs over the last 80 years revealed that the number of new scaffolds combined with molecular shape has increased over time, even though diversity of topological shapes in the set of known drugs remains low. 3 Inspection of the top 100 most frequently used rings and ring systems from small molecule drugs listed in the FDA Orange book before January 2020 1,3 did not reveal indoloquinoline or indolobenzazepine scaffolds (I−III in Chart 1).
However, rings and ring systems that the latter two classes of compounds are built upon (Chart 2) are among the top 100 rings and ring systems.
The lack of larger eight-membered azocine and ninemembered azonine rings among them is likely due to general conservatism in the design and preparation of new compounds, which are structurally similar to already approved drugs. Involvement of 3D metrics 13 such as shape or molecular electrostatic potentials in new bioactive scaffolds should increase their diversity and innovative value. Only 5% of drugs do not contain any sp 3 carbons, while 40% of drugs do not contain any sp 3 carbons in a ring. 1 At the same time, it is widely believed that introducing three dimensionality should have a positive impact on the clinical success of a potential drug. 14 Escape from flatland has been realized by us via substitution of the six-membered N-containing ring in indoloquinolines by the seven-membered azepine ring in indolobenzazepines. The effect of this structural change on antiproliferative activity and underlying mechanism of cytotoxicity has been elucidated recently. 15−17 Moreover, it was shown that the position of the sp 3 -hybridized carbon atom in a seven-membered N-containing ring has a marked effect on scaffold folding, 18 which distinctly impacts the molecular shape of the drug molecule.
The most widely used chemotherapeutics continue to be cisplatin-and platinum-based complexes, such as carboplatin and oxaliplatin. However, the use of these complexes is limited by their toxicity and acquired drug resistance. 19 Other third row transition metals, that is, osmium, iridium, and gold, 20−24 as well as second row transition metals, that is, ruthenium and rhodium, [21][22][23]25,26 were reported to be suitable for the development of potential anticancer drugs. The serious toxicity of the Pt-based drugs has stimulated extensive search for biologically essential metals and, in particular, of those of the first row, which play important biological roles in living organisms, in order to improve the pharmacological properties and reduce the general toxicity of the potential anticancer drugs. One of these metals, which attracted much attention over the last few years is copper. 23,27 Herein, we report on the synthesis and full characterization of a series of Schiff bases HL 1 −HL 5 and their ruthenium(II), osmium(II), and copper(II) complexes 1a, 1b, and 2−5 in Scheme 1 based on new four ring fused systems, which are called indolo [3,2-e]benzazocines and contain an eightmembered azocine ring with two sp 3 carbons in it, and evaluation of their anticancer potential. In particular, their cytotoxicity in a panel of human cancer cells including Chart 1. 7H-indolo [3,2-c]quinoline-6(5H)-one (I), 7,12-dihydroindolo [3,2-d] [1]benzazepine-6(5H)-one (II), 5,8dihydroindolo [3,2-d] [2]benzazepine-7(6H)-one (III), and 5,6,7,9-tetrahydro-8H-indolo [3,2-e]benzazocin-8-one (IV). Chart 2. Ring and ring systems involved in indoloquinoline and indolobenzazepine scaffolds, which are among the top 100 most frequently used ring systems from small molecule drugs sorted by descending frequency. Scheme 1. Synthesis of Ruthenium(II) and Osmium(II) Complexes 1a and 1b as Well as Copper(II) Complexes 2−5 from Indolobenzazocine-Derived Ligands HL 1 −HL 5 ; The Underlined Number Indicates the Compound Studied by SC-XRD multidrug-resistant cell lines (LS-174 and A549) obtained from solid tumors in 2D and 3D culture cells, the ability to disturb cell cycle progression and apoptosis/necrosis induction, and kinase inhibition potential were investigated. Their performance was compared with that of previously reported ring systems featuring a six-membered flat and a seven-membered folded N-containing rings, namely, indolo- [3,2- −S26), while that of 2−5 was proven by elemental analysis. In addition, the purity of HL 5 and 5 was confirmed by HPLC coupled with high-resolution ESI mass spectrometry (HR ESI MS) ( Figures S27 and S28). Finally, the coordination geometry and the molecular shape of 3 were established by SC-XRD analysis.
X-ray Crystallography. The result of SC-XRD study of complex [CuCl 2 (HL 3 )]·3MeOH (3) is shown in Figure 1, with pertinent bond distances (Å), bond angles, and torsion angles (deg) quoted in the legend. Details of data collection and refinement are collected in Table S1. The complex crystallized in the monoclinic space group P2 1 /c with one molecule of the complex and three molecules of methanol in the asymmetric unit. The copper(II) complex is fivecoordinate square-pyramidal (τ 5 = 0.14) 29 with nitrogen atoms N7, N15, and N18 and chlorido coligand Cl1 occupying the four sites of pyramide basis and another chlorido coligand Cl2 in the apical position. The bond lengths Cu−N7, Cu− N15, Cu−N18, and Cu−Cl1 (see legend to  Figure S29 in the Supporting Information.

Stability of Potential Ligands and Metal Complexes in Aqueous
Solutions. The stability of ligands HL 2 and HL 3 in aqueous solution containing 1% DMSO over 24 and 48 h was monitored by UV−vis spectroscopy ( Figure S30 in the Supporting Information). Similarly, the behavior of complexes 1a and 1b as well as 2 and 3 was investigated in aqueous solutions containing 1% DMSO over 12 and 48 h by optical spectroscopy (Figures S31 and S32). All compounds studied remained intact as no changes in the UV−vis absorption spectra over 48 h were observed. High thermodynamic and kinetic stability of five-coordinate complexes reported herein can presumably be explained in terms of hard and soft acids and bases theory. Cu 2+ as a borderline metal ion is expected to bind to a soft base, for example, S atom instead of terminal N atom, as is the case for thiosemicarbazides. However, the hard−soft character of the metal ion might be altered by the other ligands attached due to symbiotic effects. 30 This might be the case herein, where bonding of copper(II) to four hard base ligands, namely, to two N atoms (N7 and N15 in Figure  1) and two Cl − co-ligands, as well as to one borderline base In addition to UV−vis data, the stability and purity of ligand HL 5 and complex 5 were tested by analytical HPLC-HR ESI MS using methanol or acetonitrile with 0.1% formic acid as the eluent over 10 min. A single peak at around 1 min corresponding to [HL 5 + H] + (found m/z = 460.0966 ( Figure  S27), calcd m/z for C 24 H 20 BrN 5 460.0975) as well as to [Cu II (HL 5 )] + (found m/z = 521.0084 ( Figure S28), calcd m/z for C 24 H 19 BrCuN 5 521.0097) was registered in agreement with other experiments. Moreover, the 1 H NMR spectra of HL 5 in 2:1 DMSO-d 6 /D 2 O at 25°C measured immediately after dissolution, 2 h later, and after 20 h did not show any changes attesting their stability in aqueous DMSO solution ( Figure  S33).
Antiproliferative Activity. The cytotoxicity of metal-free indolobenzazocines HL 1 −HL 5 and their metal complexes 1a, 1b, and 2−5 was investigated by the colorimetric MTT assay in a panel of four human cancer cell lines, namely, human colon carcinoma HCT116 and LS-174, breast adenocarcinoma MDA-MB-361, and lung adenocarcinoma A549, as well as in human non-malignant cell line MRC-5 maintained as monolayer culture. Cisplatin or cis-diamminedichloridoplatinum(II) (CDDP), a well-known chemotherapeutic agent, was used as a positive control. The results obtained after 72 h of continuous drug action are presented as IC 50 values (μM) in Table 1.
For comparison, the IC 50 values for related complexes, in which the eight-membered azocine ring is replaced by a sevenmembered azepine ring (A and C) or by a six-membered pyridine ring (B and D) (Chart 3) in A549 cells, are also included in Table 1.
The data collected in Table 1 indicate that indolobenzazocines HL 2 −HL 5 show high cytotoxic activity with IC 50 values in the low micromolar range and are significantly more efficient than HL 1 and the reference chemotherapeutic drug cisplatin.
Modification of the original indolobenzazocine scaffold at the lactam moiety has a huge favorable effect on antiproliferative activity compared to modification performed at position 10 of indole moiety, a structure−activity relationship also noticed for paullones. 33 Coordination of HL 1 to ruthenium(II)-arene and osmium(II)-arene as well as HL 3 and HL 5 to copper(II) enhanced their cytotoxicity, while binding of HL 2 and HL 4 to copper(II) did not result in an increase of their antiproliferative activity. As can be seen from Table 1, the most active were complexes 3 and 5, both showing IC 50 values in the low micromolar concentration range. Importantly, 5 was more selective for HCT116 cells than for non-tumor MRC-5 cells (selectivity index 2.1). Cell survival diagrams for the two lead drug candidates 3 and 5 are shown in Figure 2. Both exhibited significantly higher antiproliferative activity compared to cisplatin in all tested tumor cells. In addition, they showed a significant effect in two multidrug-resistant cell lines (LS-174 and A549).
In terms of selectivity for tumor cell lines, HCT116 (colorectal carcinoma) was the most chemosensitive line. The cytotoxic potential of ligands HL 1 −HL 5 in HCT116 cells follows the order HL 5 > HL 4 > HL 3 > HL 2 > HL 1 , which closely correlates to the order of cytotoxic activity of the metal complexes: 5 ∼ 3 > 4 > 2 > 1b > 1a. Analysis of structure− activity relationships indicates that a methyl substituent at the Schiff base C�N group (R 2 = CH 3 ) highly contributes to enhancement of antiproliferative activity of ligands HL 3 and HL 5 and the corresponding complexes 3 and 5, while the presence of bromide R 1 = Br at indole moiety at position 10 (R 1 = Br in Scheme 1, see also Figure 1) had a smaller but favorable effect on the biological activity of both HL 5 and 5.
In addition, comparison with ruthenium and osmium complexes with a related paullone (Chart 3, A and Table 1) shows that replacement of eight-membered azocine ring by seven-membered azepine has a strongly favorable effect on antiproliferative activity, irrespective of the metal ion. 31 On the    To determine whether the suppression of cancer cell growth by investigated agents was associated with a cell cycle arrest, flow cytometry analysis of the DNA content was performed in HCT116 and LS-174 cells by propidium iodide (PI) staining. Cells were treated with IC 50 and 3× IC 50 concentrations of HL 5 and 5 or 10 μM cisplatin for 24 and 48 h.
Both compounds HL 5 and 5 showed dose-and timedependent effects on cell cycle progression in HCT116 and LS-174 cells. As shown in Figure 3, upon exposure of the HCT116 cells to HL 5 and 5, cell cycle phase distribution has not considerably changed over the first 24 h, when compared to the non-treated cell population. However, after prolonged 48 h action, ligand HL 5 and complex 5 showed a similar trend causing subtle dose-dependent arrest in both S and G2M phases, while sub-G1 peak, which is considered as hallmark of internucleosomal DNA cleavage, 34,35 was not detected.
Analysis in colon carcinoma LS-174 cells revealed that both ligand HL 5 and complex 5 showed time-and concentrationdependent perturbations of cell cycle, which at a lower time point (24 h) were characterized by transient arrest in the S phase, indicating stalled DNA replication. With prolonged treatment (48 h), ligand HL 5 and complex 5 induced further perturbations of cell cycle with an increase of sub-G1 population, reaching up to 12.31% of all events. We may conclude that different cell cycle perturbations following action of HL 5 and 5 in colon carcinoma HCT116 and LS-174 cells are induced due to the different kinetics and different modes of cell death induction.
Cisplatin as a reference compound and a typical DNAdamaging drug impaired progression in the S phase at 24 h time point, followed by G2M arrest at 48 h, as the result of the formation of cisplatin DNA adducts and blockage of DNA replication. 36−38 Annexin V-FITC Apoptosis Assay. The potential of HL 5 and 5 to induce apoptosis was analyzed after 24 or 48 h of treatment with IC 50 and 3× IC 50 concentrations by flow cytometry, following Annexin V-FITC/propidium iodide dual staining and compared to that of cisplatin. The obtained experimental data are presented in Figure 4 as percentages of early apoptotic cells (Annexin V-positive/PI-negative staining), late apoptotic and necrotic cells (Annexin V-positive/PIpositive staining), and dead cells (Annexin V-negative/PIpositive staining). By the current test (BD Pharmingen protocol) 39 for the cells which are already dead (Annexin Vnegative/PI-positive staining), we cannot distinguish between types of the occurred cell death. Dot plot diagrams are shown in Figure S34.
In HCT116 cells, after 24 h of treatment, only cisplatin induced a small increase in percentage of early apoptotic cells. Prolonged incubation (48 h) led to an exponential increase of the number of the early apoptotic cells (Annexin V-positive/ PI-negative staining). Early apoptosis staining at the highest concentrations of agents (3× IC 50 ) reached 19.14% in cisplatin-treated cells, 19.58% in HL 5 -treated cells, and 11.83% in cells treated with complex 5 versus 2.29% of the non-treated control. A lower number of late apoptotic/necrotic (Annexin V-positive/PI-positive staining) or dead cells (Annexin V-negative/PI-positive staining) was detected. This result was in line with the perturbations of cell cycle occurred after prolonged 48 h action of both complex 5 and HL 5 in HCT116, where sub-G1 peak, which is a marker of DNA cleavage, an event characteristic for late apoptotic (or even necrotic) changes, 35,38,40 was not detected. Apoptosis is a dynamic and kinetic event that can be affected by the cell type, apoptotic inducer, and cell cycle. It is also of note that the Annexin V-positive/PI-negative staining occurs in the early phase of apoptosis, when DNA fragments are not yet detected. 35,38,40 In LS-174 cells after 24 h of treatment, complex 5 showed dose-dependent behavior and induced apoptosis at IC 50 (up to 7.26 vs 1.01% for control cells) as well as considerable cell death at a higher dose, 3× IC 50 (up to 22.86 vs 1.63% in control cells). After prolonged treatment (48 h) with HL 5 or 5, as presented in Figure 4, a substantial increase of the number of dead cells with a disturbed cell membrane (Annexin Vnegative/PI-positive) was observed. However, we cannot distinguish dead cells that have undergone apoptotic death from those that have died as a result of a necrotic pathway. 39 At the highest concentrations of agents (3× IC 50 ), both HL 5  (1500 c/w) cells were seeded into a low-attachment U96-well plate Thermo Scientific Nunclon Sphera. After 4 days of culture (spheroidization time), the MCTSs pre-selected for a homogeneous volume and shape were treated with HL 5 , 5, and CDDP. Bright-field images were obtained using an inverted microscope.

Inorganic Chemistry
pubs.acs.org/IC Article appearance of enlarged individual cells with long pseudopods, compared with control LS-174 cells that normally grow in islands. These observations are compatible with our results of the cell cycle analysis which showed the potential of tested compounds to affect cell division and induce cell cycle arrest, resulting in enlarged cells. Moreover, the presence of floating rounded and irregularly shaped cells indicates that ligand HL 5 and complex 5 caused disruption of molecular mechanisms leading to cell death, which were demonstrated in apoptosis study by Annexin V-FITC binding. Antiproliferative Activity of the Investigated Agents in 3D Multicellular Tumor Spheroid Model. Due to the complex tissue environment, 3D multicellular tumor spheroid (MCTS) models mimic the in vivo architecture closer than 2D cell cultures, thereby allowing more precise prediction of effectiveness of organic drug candidates as well as metal-based ones in animal models. 41−44 The efficacy of HL 5 , 5, and cisplatin in the 3D MCTS model was investigated in two human colorectal cancer cell lines (CRC) LS-174 and HCT116. We used ultra-low attachment (ULA) plates for the growth of spheroids. Spheroids were incubated for 72 h and then photographed, and IC 50 values were determined using MTT assay. Tumor spheroids of diameter >500 μm selected for treatment reflect to a certain extent the tumor complexity as they are composed of several specialized areas and layers where cells show different phenotypic, functional, and metabolic behaviors. 45 They display an organized architecture with an external layer composed of proliferating cells, an intermediate zone composed of quiescent and senescent cells, and an inner apoptotic and necrotic core which is the result of the reduced distribution of nutrients and oxygen in these areas. 46,47 Analysis of growth inhibition images after 72 h of drug treatment of HCT116 cells with different drug concentrations showed that HL 5 induced growth inhibition of spheroids in a concentration-dependent manner. Complex 5 also induced growth inhibition with its apparent effect at 5 μM concentration, while higher concentrations of 10 and 20 μM seem to induce weakening of contacts between cells and loss of compactness of spheroids. The architecture of spheroids is lost as the external proliferative layer of cells (rim) disappears and the number of dying cells increases. Treatment with CDDP induced growth inhibition of spheroids in a concentrationdependent manner with an effect similar to complex 5, characterized by losing the spheroid architecture and decay of an external proliferative layer (rim) at higher concentrations up to 20 μM. Analysis of LS-174 spheroids after treatment did not show evident inhibition of growth/size of spheroids as was the case with HCT116 cells but showed loss of architecture of spheroids in terms of compactness decay of the external proliferative layer at higher drug concentrations.
In agreement with the images obtained ( Figure 6), IC 50 values determined by MTT assay in the 3D culture model of MCTS presented in Table 2 revealed a lower cytotoxic effect of HL 5 and 5 than obtained in the 2D model. However, the important level of activity, higher than the activity of cisplatin, being below 10 μM for 5 was determined on both cell lines. In HCT116 cells, HL 5 showed approximately 10× higher IC 50 value in 3D versus 2D model, with IC 50 values being 8.80 and 0.9 μM, respectively. Complex 5 showed approximately 3× higher IC 50 value in 3D versus 2D model in HCT116 cells, with IC 50 being 2.28 μM and 0.8 μM, respectively. These results indicate that complex 5 retained its cytotoxic potential in the 3D models of colorectal cancer more than its corresponding ligand HL 5 . In LS-174 cells, ligand HL 5 showed an IC 50 value over 20 μM in the 3D model and IC 50 = 3.6 μM in the 2D model. In turn, complex 5 showed 4× higher IC 50 value in 3D versus 2D model, with IC 50 being 9.3 μM and 2.1 μM, respectively. Cisplatin maintained nearly the same cytotoxic potential in the monolayer or MCTS model in both cell lines, with IC 50 values being in accordance with the literature data. 34,48 Complex 5 exhibited superior cytotoxic activity compared to cisplatin in both cell models. We may conclude that HCT116 cells were particularly sensitive to the action of complex 5 in both cell models. MCTS represents a valuable tool for in vitro drug investigation as an extrapolation to conditions in vivo (such as gradient of nutrients and oxygen, cell−cell and cell−extracellular matrix interactions, etc.), which affect drug efficiency and determine tumor cell susceptibility/ resistance to drug action. 45 Enzyme Inhibition. Paullones were first reported as ATPcompetitive CDK1, CDK2, CDK5, and GSK-3β inhibitors. 49,50 In vitro assays in a panel of 28 enzymes performed several years later 51 confirmed the selectivity of Kenpaullone to GSK-3β and CDK2 with IC 50 values 0.23 and 0.67 μM, respectively, but, in addition, Lck, a member of Src family of protein kinases, was inhibited with similar potency (IC 50 0.47 μM for both Kenpaullone and Alsterpaullone). A further update reported in 2007 for a panel of 80 kinases provided further evidence of high selectivity of Alsterpaullone and Kenpaullone for GSK-3β and CDK2. 52 Taking into account all these data, the ability of HL 5 and 5 to disturb cell cycle progression, and the close similarity of core structures in paullones and in our current lead species HL 5 and 5, we decided to test the inhibitory potency of the latter two compounds against 7 particular enzymes from 50 currently available at the Kinase Centre of the University of Dundee, namely, CDK2, CDK5 and CDK9, GSK-3α, GSK-3β, Lck, and Src. It is also worth noting that the recently reported indolo[2,3-c]quinolinederived compound and its copper(II) complex, 53 whose main organic scaffold differs from that in HL 5 and 5 more significantly, namely, by the flip of indole moiety and planarity of the core structure, showed a quite different kinase inhibitory pattern. The lead organic compound revealed good potency against PIM-1, while the copper(II) complex showed significant inhibition of the activity of SGK-1, PKA, CaMK-1, GSK-3β, and MSK1 from a panel of 50 kinases.
By using a cell-free radioactive filter binding assay, the inhibitory activity of the two lead drug candidates was assessed, and the data are summarized in Table 3 and Figure S35. Both HL 5 and 5 effectively inhibited all tested kinases, but Cdk9, with IC 50 values in the low micromolar concentration range. Generally, complex 5 revealed 2 to 4× higher efficacy of inhibition than metal-free ligand HL 5 . Complex 5 demonstrated the most effective inhibition against GSK-3α and Lck. The activity of novel agents against several kinases can be both an advantage and a disadvantage, as discussed in more details in the review articles. 54−56 Briefly, to avoid unpredictable toxic effects, research efforts are focused on design of highly selective inhibitors, but tumor cell survival and progression is a multifactorial process, sustained by a complex network of protein kinases and cross-talk among different signaling pathways, so it seems reasonable to establish anticancer therapies that target several kinases associated with tumor growth. Overall, the collected data suggest that HL 5 and 5 are potentially multi-kinase inhibitors.
To find out the physiological role(s) of GSK-3α and Lck in cell-based assays, the same effect should be observed with at least two structurally unrelated inhibitors of these protein kinases. 57 In accord with the results reported previously, 51 the combined use of 5 and LiCl may help in identifying the substrates and physiological roles of GSK-3α in cells. This kind of investigations is imperative and will be performed in the future.

■ CONCLUSIONS
In summary, in addition to bioactive ring systems documented in the literature, such as indolo [3,2-

c]quinolines and indolo-[3,2-d][1]benzazepines and indolo[3,2-d][2]benzazepines in-
corporating either a six-or seven-membered N-containing ring, respectively, an entry to indolo[3,2-e]benzazocine scaffold containing an eight-membered azocine ring has been realized. Five bi-and tridentate ligands (HL 1 −HL 5 ) and ruthenium(II) (1a), osmium(II) (1b), and copper(II) complexes (2−5) have been synthesized and characterized. In vitro cytotoxicity tests against four cancer cell lines (LS-174, HCT116, MDA-MB-361, and A549) revealed ligand HL 5 and copper(II) complex 5 as series leaders, with a strong cytostatic effect higher than that of cisplatin (particularly against human colorectal cancer cell lines HCT116 and LS-174). These compounds were also more selective for HCT116 cells than for normal human lung fibroblasts (MRC-5). Morphological studies in the presence of HL 5 and 5 showed disturbance of cell growth over time. In addition, cell cycle analysis revealed that both HL 5 and 5 induce concentration-and time-dependent arrest of cell cycle phases, which differs from that for cisplatin. Annexin V-FITC apoptosis assay showed dominant apoptosis inducing potential after prolonged treatment (48 h) in HCT116 cells.
HL 5 and 5 can inhibit Cdks, as further supported by cell-free radioactive filter binding assay against Cdk2 and Cdk5. However, human cells integrate mitogen and stress stimuli before committing to the cell cycle by regulating the levels of different Cdks, which also play roles in transcriptional processes and apoptosis programs. Thus, due to the complex and multiple roles of Cdks in cell signaling, additional studies are needed to precisely address the molecular mechanism underlying the Cdk-inhibitory and cytotoxic action of the tested compounds. In general, Cdk protein kinases share structural and functional similarities, and development of the small molecule Cdk inhibitors with distinct specificity is an extremely challenging task. 58,59 As for paullones, GSK-3α, GSK-3β, Src, and Lck are among other possible targets for lead drug candidates HL 5 and 5. Complex 5 showed higher antiproliferative activity in the 3D culture model of MCTS than clinical drug cisplatin, attesting its suitability for in vivo assays. Thus, these findings indicate that incorporation of an eight-membered azocine ring into related paullone structures enriches the available chemical space of bioactive scaffolds, and, in combination with substitution at the lactam unit, Schiff base C�N bond and bromination at position 10 are effective tools for fine-tuning the biological potency of copper(II)-based anticancer drugs. ■ EXPERIMENTAL SECTION General Information. NMR spectra were recorded on Bruker AV700, AV600, or AV500 spectrometers. 1 H and 13 C NMR chemical shifts (δ) are given in ppm relative to TMS using the residual solvent signals as references and converting the chemical shifts to the TMS scale. ESI-MS spectra were recorded on a Bruker amaZon speed ETD spectrometer (3D-ion trap). High-resolution ESI mass spectra were recorded on a Bruker maXis UHR-TOF spectrometer.
Materials. 5-Nitro-1H-indole-3-carboxaldehyde, 2-iodobenzonitrile, and silver(I) carbonate were purchased from abcr. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI·HCl) and trifluoroacetic acid were purchased from IRIS biotech, while palladium (10%) on activated charcoal, tert-butyl dicarbonate (Boc 2 O), palladium(II) acetate, 4-(dimethylamino)pyridine (DMAP), sodium chlorite, and sulfamic acid were obtained from Sigma-Aldrich. 2-Formylpyridine, absolute dimethylformamide (DMF), tetra-n-butylammonium fluoride (TBAF), 4-toluenesulfonyl chloride, triethylamine, sodium bicarbonate, magnesium sulfate, and borane solution (1 M in THF) were bought from Fisher/Acros Organics. Triphenylphosphine, sodium bicarbonate, and celite were purchased from Alfa Aesar. 2-Iodobenzeneethylamine was prepared by using a literature protolol. 28 Synthesis of the main organic scaffolds b 1 −h 3 is described in detail in the Supporting Information. 1 H NMR spectra of intermediate Synthesis of Ligands HL 1 −HL 5 . HL 1 ·0.5CH 3 OH: To a solution of g 1 (520 mg, 1.88 mmol) in anoxic ethanol (80 mL) in a 250 mL Schlenk tube, 2-formylpyridine (196 μL, 2.06 mmol) was added, and the mixture was stirred at 85°C for 20 h. On the next day, the reaction mixture was cooled to room temperature and the solvent was evaporated under reduced pressure. The product was crystallized in methanol and isolated as a yellow powder. Yield: 380 mg, 55%. 1 , 1.31 mmol), and the solution was stirred at 75°C overnight. On the next day, the reaction mixture was cooled to room temperature and the yellow precipitate was filtered off. Yield: 345 mg, 72%. 1 , 0.45 mmol), and the mixture was stirred at 75°C overnight. On the next day, the reaction mixture was cooled to room temperature and concentrated under reduced pressure. The yellow product was precipitated by addition of diethyl ether (5 mL) and filtered off. Yield: 171 mg, 85%. 1   )] + 477.08. X-ray diffraction quality single crystals were obtained by slow evaporation of a methanolic solution of 3. 4·CH 3 OH: To a solution of HL 4 (20 mg, 0.05 mmol) in 2propanol (6 mL), a solution of CuCl 2 ·2H 2 O (8 mg, 0.05 mmol) in methanol (1 mL) was added. The reaction mixture was heated to reflux for 15 min, cooled down, and allowed to stand at room temperature overnight. The product was filtered off and dried in vacuo to give a green powder. Yield: 20 mg, 78%. Anal. Calcd for C 23  Crystallographic Structure Determination. The measurements were performed on a Bruker X8 APEXII CCD and Bruker D8 Venture diffractometers. Single crystals were positioned at 27, 60, and 60 mm from the detector, and 500, 1000, and 9214 frames were measured, each for 8, 1, and 1 s over −0.360, −0.360, and 0.360°scan width for f 2 ·CH 2 Cl 2 , g 2 ·CH 2 Cl 2 , and 3·3MeOH, respectively. The data were processed using SAINT software. 60 Crystal data, data collection parameters, and structure refinement details are given in Table S1. The structures were solved by direct methods and refined by full-matrix least-squares techniques. Non-H atoms were refined with anisotropic displacement parameters. H atoms were inserted in calculated positions and refined with a riding model. The following computer programs and hardware were used: structure solution, SHELXS and refinement, SHELXL; 61 molecular diagrams, ORTEP; 62 computer, Intel CoreDuo. CCDC 2194805 (f 2 ·CH 2 Cl 2 ), 2194806 (g 2 ·CH 2 Cl 2 ) and 2194807 (3·3MeOH).